During cell division daughter cells must receive one and only one copy of each and every chromosome. To achieve this all chromosomes must be duplicated and the duplicated products must be recognized as being equivalent, i.e. sister chromatids, so that they can be segregated to the two daughter cells. The identity of the sister chromatids is maintained by the presence of sister chromatid cohesion, which is dissolved only after all duplicated chromosomes have formed bipolar spindle attachments. A key regulator of this process in budding yeast is the protein Pds1. Functional homologues of Pds1, known as securins, are found in many organisms including human, where securin over expression is linked to genomic instability. Pds1 inhibits anaphase initiation by inactivating a conserved protease known as separase (Esp1 in budding yeast) that dissolves cohesion between sister chromatids. Esp1 becomes active only after Pds1 is degraded in a process that involves a ubiquitin ligase called the anaphase promoting complex/cyclosome (APC/C). During Pds1 degradation the APC/C acts in conjunction with an associated subunit, the Cdc20 protein. We have previously shown that Cdc20 acts as a substrate recognition subunit of the APC/C and that it binds Pds1 directly. Pds1 function becomes crucial in the presence of DNA or spindle damage, when it inhibits mitotic progression until the damage is repaired. In the presence of DNA damage Pds1 is stabilized and we have recently determined the molecular mechanism for this stabilization. In addition to its role as an inhibitor of Esp1, Pds1 is also required for proper Esp1 function. In the past year we carried out two genetic screens that exploited these properties of Pds1 and that have lead to the identification of proteins involved in spindle function, DNA integrity and Esp1 activation (section 1). We also discovered a novel role of Esp1 in regulating nuclear positioning during mitosis (section 2). Finally, we are searching for proteins that are needed for nuclear structure and integrity. Thus far we have focused on two proteins, Mlp1 and Mlp2, and found that they are needed for chromosome integrity (section 3). 1. The role of Pds1 as a mitotic regulator. Pds1 is not essential for viability but there are several conditions under which cells lacking Pds1 function cannot survive. These include conditions that lead to spindle defects or induce DNA damage. In addition, we hypothesize that Pds1-independent defects in the activation of Esp1 or nuclear localization will also render Pds1 essential because under these conditions cells will have to depend on the ability of Pds1 to promote Esp1 activation. To identify proteins required for spindle function, DNA integrity or Esp1 activation we carried out two genetic screens for mutants whose viability depends on Pds1 (known as a yeast synthetic lethality screen). One of the genes identified was CDH1. The Cdh1 protein is required for cyclin degradation. We found that the cdh1 mutant strain requires the spindle checkpoint, and Pds1 in particular, to prevent chromosome mis-segregation. These and additional results suggest that an untimely increase in cyclin-Cdk activity can lead to genomic instability (published in Genetics, 2003). We also conducted a genetic screen in collaboration with Dr. Mike Tyers (Samuel Lunenfeld Research Institute, Toronto Canada), in which a deletion of Pds1 was combined, individually, with a collection of strains each carrying a deletion of one out of the 4600 non-essential genes in budding yeast. This screen has yielded over twenty mutants that require Pds1 for viability, many of which were not previously known to be involved in cell cycle regulation. These genes were analyzed for possible involvement in spindle function, DNA replication/damage and Esp1 inactivation. As expected, several of the mutants were indeed defective in spindle function, one required the DNA damage checkpoint for viability and two were found to be involved in DNA replication. Interestingly, in the course of this study we discovered two heat shock/chaperon proteins needed for Esp1 activation. A manuscript describing these findings has been accepted for publication in Genetics. 2. Esp1 and nuclear positioning. In budding yeast, cells lacking separase/Esp1 function exit mitosis with an undivided nucleus localized to the daughter cell. We found that this daughter preference was not due to the requirement for Esp1 in separation of sister chromatids. Rather, Esp1 affected nuclear positioning as part of the Cdc14 Early Anaphase Release (FEAR) pathway, which was previously shown to be needed for mitotic exit. We found that the nuclear segregation defect in FEAR mutants did not stem from non-functional spindle poles or the absence of cytoplasmic microtubules, and we hypothesize that the FEAR pathway regulates one or more microtubule associated motor proteins. We propose that at anaphase onset the FEAR pathway activates cytoplasmic microtubule-associated forces that facilitate chromosome segregation to the mother cell. This study was recently published in Developmental Cell. 3. Exploring nuclear structure and integrity. Many nuclear processes, including transcription, DNA replication and gene silencing, are functionally linked to nuclear structure. In an attempt to uncover proteins that contribute to nuclear structure and integrity focused on the Mlp1 and Mlp2 proteins that were reported to be required for proper intranuclear chromosome localization. We found that the absence of these proteins leads to the activation of the DNA damage checkpoint pathway and elongated telomeres. Moreover, we found that these proteins are important for chromosome integrity and in their absence the rate of chromosomes rearrangement is significantly increased. We hypothesize that Mlp1 and Mlp2 are involved in DNA metabolism related processes, possibly by providing the appropriate environment at the nuclear periphery that is needed to ensure proper chromosome structure. A manuscript describing these findings has been submitted for publication.